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This study was carried out to screen for antimicrobial activities against common pathogenic and food spoilage bacteria, yeasts and molds, toxinogenic potential and sensitivity to antibiotics of 26 Bacillus strains (11 Bacillus subtilis, 5 Bacillus megaterium, 4 Bacillus amyloliquefaciens, 4 Bacillus pumilus and 2 Bacillus thuringiensis) isolated from different samples of Hibiscus sabdariffa fermented seeds (Mbuja). Antimicrobial assays were realised according to the cross-streak and the overlay methods. The main toxin and bacteriocin encoding genes were screened by PCR using specific primers and antibiotic resistance was assessed by the disc diffusion method. Bacillus species showed variable ability to inhibit bacterial and/or fungal species. The most antibacterial strains were B. amyloliquefaciens (S1 and S5) and B. subtilis (S12) whereas the most antifungal ones were B. megaterium S8 and S9. Subtilin and subtilosin A genes were detected in seven strains of B. subtilis but they were not associated with antimicrobial activities. All strains, except B. thuringiensis, did not contain toxin encoding genes and were sensitive to most tested antibiotics. This suggests that antimicrobial strains could be used in starter cultures for a controlled fermentation to produce Mbuja in order to better control the fermentation process of Mbuja and to increase consumer’s safety.Keywords: Bacillus, antimicrobial activities, toxins, antibiotic resistanceAfrican Journal of Biotechnology, Vol 13(31) 3617-3627